SUMMARY In this application we propose to investigate the biological features of innate CD8? cells (or iCD8? cells), a novel lymphoid population present in the intestinal epithelium discovered by our group. A hallmark of this population is the expression of CD8??, lack of T and B cell receptors, and the production of cytokines/chemokines that indicate an innate immune phenotype. iCD8? cells are a main source of osteoponin in the intestinal epithelium, a cytokine known for its role in tissue remodeling but also for its capacity to stimulate Th1 and Th17 immune responses. Interestingly, using a strain of mice lacking iCD8? cells (E8I mice; E8I is an essential enhancer for CD8?? expression in IEL) we found that the levels of -/- osteopontin in the intestinal epithelium are decreased in comparison to wild type mice, and that E8I-/- mice have a deficiency in NKp46+NK1.1+ IEL. These and other preliminary results have led to the hypothesis that iCD8? cells, through production of factors such as osteopontin, promote the survival and maintenance of IEL. In order to pursue this hypothesis, we propose the following complementary, yet independent aims: Aim1. To determine the contribution of iCD8? cells and osteopontin to IEL maintenance and survival. In this aim we will determine: a) the impact of iCD8? cells and osteopontin in the survival and proliferation of different IEL subsets; b) the impact of iCD8? cells and osteopontin in the immune environment of the intestines; and c) the role of osteopontin ligands in IEL homeostasis. Aim 2. To define the role of iCD8? cells and osteopontin during intestinal inflammation. In this section we will determine the role of iCD8? cells and osteopontin in three different disease models: a) intestinal inflammation by anti-CD40 antibody treatment, b) Citrobacter rodentium infection, and c) inflammation caused by adoptive transfer of effector T cells into immunodeficient mice. Aim 3. To determine the mechanisms controlling IEL homeostasis by iCD8? cells. Our previous publication indicates that IL-12 provides stimulus for activation of iCD8? cells. In this section we will determine a) the impact of IL-12 in activation of iCD8? cells, with implications on OPN secretion and IEL survival, and b) we will determine the gene expression signature of IEL responding to iCD8? cell/OPN stimulation.